A SWI/SNF-dependent transcriptional regulation mediated by POU2AF2/C11orf53 at enhancer

Recent studies have identified a previously uncharacterized protein C11orf53 (now named POU2AF2/OCA-T1), which functions as a robust co-activator of POU2F3, the master transcription factor which is critical for both normal and neoplastic tuft cell identity and viability. Here, we demonstrate that POU2AF2 dictates opposing transcriptional regulation at distal enhance elements. Loss of POU2AF2 leads to an inhibition of active enhancer nearby genes, such as tuft cell identity genes, and a derepression of Polycomb-dependent poised enhancer nearby genes, which are critical for cell viability and differentiation. Mechanistically, depletion of POU2AF2 results in a global redistribution of the chromatin occupancy of the SWI/SNF complex, leading to a significant 3D genome structure change and a subsequent transcriptional reprogramming. Our genome-wide CRISPR screen further demonstrates that POU2AF2 depletion or SWI/SNF inhibition leads to a PTEN-dependent cell growth defect, highlighting a potential role of POU2AF2-SWI/SNF axis in small cell lung cancer (SCLC) pathogenesis. Additionally, pharmacological inhibition of SWI/SNF phenocopies POU2AF2 depletion in terms of gene expression alteration and cell viability decrease in SCLC-P subtype cells. Therefore, impeding POU2AF2-mediated transcriptional regulation represents a potential therapeutic approach for human SCLC therapy.

A) The Venn diagram shows the overlap of the significantly differentially expressed genes in BRG1/BRM inhibitor BRM014 treated or POU2AF2/POU2F3 depleted cells (adj.p < 0.01, |log2FC| > 0.5).Data are derived from two biological replicates.The pathway analysis with the overlapping down-regulated genes (B) and up-regulated genes (C) from (A).The -log10(P) value was calculated by Metascape software 52 .D) The RNA-seq results of the expression levels of scatter plot shows the correlation between POU2F3 and POU2AF2 peaks in NCI-H526 cells.Pearson correlation = 0.74.B) The overlapped POU2AF2 and POU2F3 peaks in NCI-H211 cells were divided into three clusters by k-means clustering based on POU2AF2, POU2F3, and histone marks (H3K4me1, H3K27ac, H3K4me3, and H3K27me3).The ChIP-seq signal of these histone marks were further centered on the three clusters.C) The scatter plot shows the correlation of gene expression change when POU2F3 or POU2AF2 were depleted by sgRNAs in NCI-H211 cells.The significantly altered genes (|log2FC| >1, adj.p < 0.01) by POU2F3/POU2AF2 depletion were highlighted in red (upregulated, n = 1138) or blue (downregulated, n = 570).Data are derived from two biological replicates.Genes with Benjamini-Hochburg adjusted p-values less than 0.01 were considered to be differentially expressed in the EdgeR analysis 49 .D) The Venn diagram shows the overlap between POU2F3 and POU2AF2 target genes in NCI-H211 cells.E) The box plot shows the significance of expression change of each cluster nearby genes upon the loss of POU2AF2 (upper panel) or POU2F3 (lower panel) with sgRNAs.p-value is calculated by twosided Wilcoxon test.Center line: median; top and bottom hinges of box: the third and first quantiles; whiskers: quartiles ± 1.5 × interquartile range.F) The track examples show the occupancy of POU2F3 and POU2AF2 at H3K27me3 and H3K4me1 occupied enhancer regions in NCI-H211 cells, and the activation of nearby gene expression upon the loss of POU2AF2.

Figure
Figure S2.POU2AF2 is essential for PRC2 maintenance and repression of Polycomb target

Figure S3 .
Figure S3.POU2AF2 interacts with the SWI/SNF complex and regulates chromatin

Figure S4 .
Figure S4.The ATPase activity of SWI/SNF complex is required for POU2AF2 mediated TAS1R3, IRAG2, AVIL, CHAT, SUCNR1, GNG13, NREP, and ASCL2 in DMSO or BRM014 (1 M) treated cells.Data are derived from two biological replicates.Source data are provided as a Source Data file.E) The bar plot heatmap shows the H3K4me1 and H3K27ac levels at Cluster 2 peaks between NCI-H526 cells transduced with either non-targeting sgRNA or two distinct POU2AF2 sgRNAs.F) The track example shows the reduction of BRG1, H3K27ac, H3K4me1 levels as well as ATAC-seq signals at POU2AF2 occupied active enhancer at CHAT gene locus.G) The average plot shows a reduction of EZH2 levels (left panel) and H3K27me3 levels (right panel) upon BRM014 treatment at Cluster 3 peaks.H) The RNA-seq results for the expression levels of FOXO1, IGFBP2, UBASH3B, and EYA1 in DMSO or BRM014 (1 M) treated cells.Data are derived from two biological replicates.Source data are provided as a Source Data file.I) The average plot shows the H3K27me3 levels at Cluster 1 and Cluster 2 peaks upon BRM014 treatment.

Figure S5 .
Figure S5.Inhibition of the ATPase activity of SWI/SNF complex as a therapeutic strategy